Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

Enhanced antitumor efficacy by blocking activation of the phosphatidylinositol 3-kinase/Akt pathway during anti-angiogenesis therapy.

Anti-angiogenesis has been a promising strategy for cancer therapy. However, many signal pathways are activated during anti-angiogenic treatment to counteract the therapeutic efficacy. Among these pathways, evidence has directly pointed to the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, whose activation resulted in tolerance to the absence of nutrients and oxygen when tumor angiogenesis has been inhibited. In the present study, we investigated the effects of blocking activation of the PI3K/Akt pathway on cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy. In modeled microenvironments in vitro, we observed that the phosphorylation of Akt in tumor cells was increased gradually in the absence of serum and oxygen in a time-dependent manner. The specific inhibitors of PI3K inhibited the proliferation of tumor cells in a dose-dependent manner in vitro. Moreover, inhibition was enhanced gradually with increased serum deprivation and/or hypoxia. In a mouse tumor model, we found the phosphorylation of Akt obviously increased following anti-angiogenic therapy using plasmids encoding soluble vascular endothelial growth factor receptor-2, but significantly reduced after treatment with LY294002. Consequently, the combinational treatment exhibited better antitumor effects compared with single treatments, presenting larger necrosis-like areas, more apoptotic cells, less microvessel density and less phosphorylated Akt in tumors. These results suggest that blocking activation of the PI3K/Akt pathway during anti-angiogenesis therapy could enhance antitumor efficacy. Thus, targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy.

[Antitumor effect by combination of shRNA interfering plasmid targeting PKM2 with recombinant endostatin].

OBJECTIVE: To investigate the enhancement effect of the combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin in the treatment of lung cancer. METHODS: Twenty five BABL/nu/nu mice bearing A549 lung cancer were divided into 5 groups (NS control, psh-Control, psh-PKM2 treated group, Endostar treated group, psh-PKM2+Endostar treated group) and treated with shRNA interfering plasmid targeting PKM2 and recombinant Endostatin respectively or in combination. The expression of PKM2 in A549 detected with immunofluorescent assay. The interference effect of psh-PKM2 was determined by Western blot. The tumor volume, microvessel density (MVD), apoptosis index (AI) and side effects were observed. RESULTS: The combination treatment of RNA interfering plasmid targeting PKM2 with recombinant Endostatin inhibited tumor growth obviously (P < 0.05); The combination group revealed a decreased MVD and an increased AI (P < 0.05). CONCLUSION: The combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin might enhance anti-tumor effect by increasing the apoptosis of the cancer cell.

[The molecular mechanism of extravasation in mouse melanoma lung metastasis model].

OBJECTIVE: To study molecular mechanisms underlying the extravasation of mice melanoma cells during lung metastasis. METHODS: B16-RED melanoma cell line was established which stably express the red fluorescent protein. B16-RED cells were compared with B16 cells in ability of proliferation and lung metastasis. A mouse lung metastasis model was established with B16-RED melanoma cells. FITC-dextran was injected i.v. and CD31 indirect immunoflourescence (IIF) staining was made to identify the location of the tumor cells and the time of tumor cell extravasation. Finally, at 48 hours post cell injection, the lung and a normal lung were removed and used for 32K mice microarray analysis. RESULTS: B16-RED was consistent with B16 in cell shape and ability of proliferation and lung metastasis. 52.7% of B16-RED melanoma cells completed the extravasation within 48 hours in mouse lung metastasis model. Many important signal pathways were involved during lung metastasis, including leukocyte transendothelial migration, MAPK signaling pathway, neuroactive ligand-receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, axon guidance, calcium signaling pathway, tight junction, etc. CONCLUSION: The extravasation during metastasis is a complex and multiple-steps process, in which many important signal pathways in host tissues were involved.

Proteomic analysis of early-response to mechanical stress in neonatal rat mandibular condylar chondrocytes.

The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four-point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 microstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2-DE and MALDI-TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling.

Antitumor and anti-angiogenesis immunity induced by CR-SEREX-identified Xenopus RHAMM.

Immunization with xenogeneic antigens is an attractive approach to induce cross-reactive humoral and cellular immunity to inhibit tumor growth or angiogenesis. To identify novel xenogenic targets for immunotherapy, we have developed a modified serological expression cloning (SEREX) strategy, termed Cross-reactive SEREX (CR-SEREX). Among 78 positive clones identified by CR-SEREX, Xenopus receptor for hyaluronic-acid-mediated motility (xRHAMM) was most frequently identified (18 times), indicating the strongest immunogenic potential for xenogenic immunotherapy. A DNA vaccine based on xRHAMM effectively induced a protective antitumor immunity against local tumor and lung metastasis in B16 melanoma mouse models. Angiogenesis was inhibited and cell apoptosis was increased within tumors. Antitumor activity of xRHAMM worked through stimulation of an antigen-specific cellular response as well as through a specific humoral response against RHAMM, as confirmed by the depletion of immune cell subsets in vivo. Thus, a xenogenic vaccine based on xRHAMM induced an effective immunity against B16 melanoma cells and endothelial cells.

Identification, characterization, and effects of Xenopus laevis PNAS-4 gene on embryonic development.

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

[Anti-tumor effects induced by the DNA vaccine coding human and mouse soluble VEGFR2].

OBJECTIVE: To develop a novel anti-angiogenesis strategy based on a DNA vaccine coding both human and mouse soluble VEGFR2. METHODS: The gene fragments coding human and mouse sVEGFR2 were amplified with PCR and cloned into pVITRO2 to generate pVITRO2-hm-sVEGFR2 recombinant. The in vitro VEGF blocking effect of the pVITRO2-hm-sVEGFR2 expression products on HUVEC cells were evaluated. The anti-tumor effect of pVITRO2-hm-sVEGFR2 was studied in mouse B16 model. The microvessels were stained by using CD31 antibody. RESULTS: The co-expressing vector pVITRO2-hm-sVEGFR2 was constructed successfully, confirmed by the restriction endonuclease digestion and sequencing. The expressing products of pVITRO2-hm-sVEGFR2 could obviously block the function of VEGF on promoting the proliferation of HUVEC in vitro. The tumor growth in mice was also significantly inhibited by pVITRO2-hm-sVEGFR2 expression. CD31 staining demonstrated that the microvessel density obviously decreased in tumor tissues treated with pVITRO2-hm-sVEGFR2. Both anti-tumor and anti-angiogenesis effects of pVITRO2-hm-sVEGFR2 were stronger than that of plasmids which coding only human or mouse sVEGFR2. CONCLUSION: pVITRO2-hm-sVEGFR2 could be a novel DNA vaccine for the anti-tumor therapy by inhibiting angiogenesis.

[IL-15 enhance the therapeutic effects of whole cell vaccine in mouse CT26 colon carcinoma model].

OBJECTIVE: To determine the enhancement effects of IL-15 to the tumor whole cell vaccine in tumor immunotherapy. METHODS: CT26 colon carcinoma model was established with BALB/c mice. Thirty two mice with CT26 colon carcinoma were divided randomly into four groups, which were subcutaneously injected at several spots with PBS (200 microL), CT26 whole cell vaccine (2 x 10(6) cells in 200 microL PBS), mIL-15 (20 microg, encapsulated with liposome in 200 microL 5% glucose solution) and CT26 whole cell vaccine (2 x 10(6) cells in 100 microL PBS) plus mIL-15 (20 microg, encapsulated with liposome in 100 microL 5% glucose solution) respectively every three days for six doses, the plasmid was injected beside the vaccine injecting spots. The size of tumors was measured every four days. All mice were sacrificed 30 days after tumor implantation. The pathologic observation and apoptotic analysis of tumors were preceded. RESULTS: CT26 whole cell vaccine combined with mIL-15 inhibited tumor growth by 45% compared with that of control group, the differences between CT26 + mIL-15 group and the other three groups were significant (P < 0. 05). The HE staining showed that the necrosis areas in tumors of the CT26 + mIL-15 group were larger than those of other three groups. The apoptotic index of tumors from the CT26 + mIL-15 group was (46.7 +/- 7.2)%, higher than that of the other three groups obviously (P < 0.05). CONCLUSION: IL-15 could enhance the therapeutic effects of CT26 whole cell vaccine in tumor immunotherapy.

A promising cancer gene therapy agent based on the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in inducing cell apoptosis during infection. To investigate whether M protein-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with cationic liposome and introduced into various tumor cell lines in vitro, including lung cancer cell LLC, A549, colon cancer cell CT26 and fibrosarcoma cell MethA. Our data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. Fifty micrograms of plasmid in a complex with 250 microg cationic liposome was injected intratumorally into mice bearing LLC or MethA tumor model every 3 days for 6 times. It was found that the tumors treated with M protein plasmid grew much more slowly, and the survival of the mice was significantly prolonged compared with the mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with M protein plasmid were even completely regressed, and the mice acquired longtime protection against the same tumor cell in rechallenge experiments. Both apoptotic cells and CD8(+) T cells were widely distributed in M protein plasmid-treated tumor tissue. Activated cytotoxic T lymphocytes (CTLs) were further detected by means of (51)Cr release assay in the spleen of the treated mice. These results showed that M protein of VSV can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effect and warrant its further development in cancer gene therapy.

Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.

A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.

RNA interference against Biot2, a novel mouse testis-specific gene, inhibits the growth of tumor cells.

Biot2 is a novel murine testis-specific gene that was first identified using the SEREX technique, and named by our laboratory. Using conventional RT-PCR and real time RT-PCR, we tested the expression profile of Biot2 in normal tissues and various murine tumor cell lines. Using RNA interference, we studied the biological function of Biot2 in tumorigenesis. We applied various types of growth assay, such as the in vitro MTT, colony-forming and BrdU incorporation assays, along with in vivo tumorigenicity assays, to reveal its inhibition of tumor cell proliferation. The results revealed that the Biot2 transcript was detected only and strongly in the testis tissues and abundantly in five types of murine cancer cell line. Treating B16 murine melanoma, LL/2 murine Lewis lung carcinoma and CT26 murine colorectal adenocarcinoma with special shRNA targeting Biot2 can significantly reduce the proliferation rate of these three tumor cell lines in vitro, as measured by the MTT, colony-forming and BrdU incorporation assays. The tumorigenicity of the CT26 cells transfected with special shRNA targeting Biot2 was also decreased distinctly in vivo compared with the control. It was therefore concluded that Biot2 plays a key role in tumorigenesis and could be a potential target for biotherapy.

[Construction and identification of eukaryotic co-expression plasmid carrying FUS1 and recombinant human IL-12 genes and its effection on apoptosis of A549 cells].

OBJECTIVE: To construct a eukaryotic co-expression plasmid containing FUS1 and IL-12, and to investigate the influence of the recombinant plasmid on the cultured human lung cancer cell line A549. METHODS: RT-PCR was applied to amplify FUS1 from MRC-5 cell, the cDNA fragment of IL-12 was derived from pORF-hIL-12 by PCR, FUS1 cDNA fragment and IL-12 cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-FUS1-IL-12. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenceing and transfected into A549 cells. The expression of genes in pVITRO2-FUS1-IL-12 were identified by RT-PCR, ELISA and Western blot. The function of pVITRO2-FUS1-IL-12 inducing the apoptosis of lung cancer was identified by Hoechst33258 and flow cytometry. RESULTS: The restriction endonuclease digestion and sequencing suggested that co-expression vector pVITRO2-FUS1-IL-12 was constructed successfully, the expression of FUS1 and IL-12 could be detected in A549 cells. The expressions of the two genes in pVITRO2 were not affected. The A549 cells transfected with pVITRO2-FUS1-IL-12 exhibited significant cell apoptosis. CONCLUSION: The recombinant eukaryotic expression vector pVITRO2-FUS1-IL-12 was constructed and expressed in eukaryotic cell successfully, which would contribute to a novel and effective treatment strategies for combined gene therapy for lung cancer.

Chronological in vivo imaging reveals endothelial inflammation prior to neutrophils accumulation and lipid deposition in HCD-fed zebrafish.

BACKGROUND AND AIMS: Hyperlipidemia-induced atherosclerosis is the major cause of heart attack and stroke in humans. However, pathological details and molecular mechanisms underlying early atherogenesis remain incompletely characterized. This study explored the early events of atherogenesis in a hypercholesterolemic zebrafish model in vivo. METHODS: We used transparent transgenic zebrafish larvae Tg(lysc:EGFP), Tg(mpx:EGFP), Tg(mpeg1:EGFP), Tg(flk1:EGFP) or Tg(lysc:EGFP/flk1:mCherry), together with fluorescently labeled control and high cholesterol diets (HCD), to dynamically investigate the early development of atherosclerosis with confocal in vivo. Endothelial cells with green fluorescence were sorted by fluorescence-activated cell sorting (FACS) to detect gene expression. Moreover, we treated hypercholesterolemic zebrafish model in vivo or human umbilical vein endothelial cells (HUVEC) in vitro with rosiglitazone, an agonist of peroxisome proliferator-activated receptor γ (PPARγ). RESULTS: We found that HCD-induced endothelial inflammation was an earlier pathological alteration than myeloid cells/neutrophils accumulation and lipid deposition in zebrafish vascular vessels of HCD-fed zebrafish. Endothelial inflammation was characterized by down-regulation of anti-inflammatory PPARγ and upregulation of pro-inflammatory tumor necrosis factor α (TNF-α) and interleukin-1ß (IL-1ß). Pharmacological treatment with rosiglitazone reversed the decrease in the expression of PPARγ and decreased expression of TNF-α and IL-1ß in HCD-fed zebrafish. Moreover, rosiglitazone ameliorated myeloid cells accumulation and lipid deposition in HCD-fed zebrafish in vivo. CONCLUSIONS: Hyperlipidemia-induced endothelial inflammation happens earlier than myeloid cell neutrophils accumulation in vascular vessels, and neutrophils accumulation is prior to lipid deposition during the initial stage of atherosclerosis. Early alleviation of inflammation induced by HCD would have a prophylactic effect for the initial development of atherosclerosis.

Liposomal honokiol, a promising agent for treatment of cisplatin-resistant human ovarian cancer.

PURPOSE: Honokiol has been receiving attention as an anticancer agent because of its anti-tumor effect. In the current study, we encapsulated honokiol with liposome and tested it on cisplatin-sensitive (A2780s) and -resistant (A2780cp) human ovarian cancer models. METHODS: The anti-tumor activity of liposomal honokiol (Lipo-HNK) was evaluated in nude mice bearing A2780s and A2780cp s.c. tumors. Mice were treated twice weekly with i.v. administration of Lipo-HNK (10 mg/kg), control liposome (10 mg/kg), 0.9% NaCl solution or weekly with intraperitoneally administered cisplatin (5 mg/kg) for 3 weeks. Tumor volume and survival time were observed. Assessment of apoptotic cells by TUNEL assay was conducted in tumor tissue. Microvessel density within tumor tissue was determined by CD34 immunohistochemistry. For in vitro study, induction of apoptosis by Lipo-HNK was examined by PI staining fluorescence microscopy, DNA fragmentation assay and flow cytometric analysis. RESULTS: Administration of Lipo-HNK resulted in significant inhibition (84-88% maximum inhibition relative to controls) in the growth of A2780s and A2780cp tumor xenografts and prolonged the survival of the treated mice. These anti-tumor responses were associated with marked increases in tumor apoptosis, and reductions in intratumoral microvessel density. CONCLUSIONS: The present findings suggest that Lipo-HNK may provide an effective approach to inhibit tumor growth in both cisplatin sensitive and -resistant human ovarian cancer with minimal side effects.

Synergistic antitumor effect of CXCL10 with hyperthermia.

PURPOSE: IFN-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) has been described as an antiangiogenic chemokine and displays a potent antitumor activity in vivo. In the present study, we try to investigate whether the combination therapy of hyperthermia, a physical antiangiogenic modality, with CXCL10 would completely eradicate the established solid tumors. METHODS: Immunocompetent BALB/c mice bearing Meth A fibrosarcoma were established. Mice were treated with either CXCL10 at 25 microg/kg once a day for 20 days, hyperthermia was given twice (at 42 degrees C for 1 h, on day 6 and 12 after the initiation of CXCL10), or together. Tumor volume and survival time were observed. The microvessel density was determined by CD31 immunofluorescence. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. RESULTS: The results showed that CXCL10 and hyperthermia inhibited the growth of Meth A fibrosarcoma and interestingly, the combination therapy enhanced the antiangiogenic effects and completely eradicated the established solid tumors. Histological examination revealed that CXCL10 + hyperthermia led to increased induction of apoptosis, tumor necrosis, and elevated lymphocyte infiltration compared with the controls. Moreover, the tumor eradicated animals developed a protective T-cell-dependent antitumor memory response against Meth A tumor cells rechallenge. CONCLUSIONS: Our finding is that the combination therapy can achieve a synergistic antitumor efficacy, supporting the idea that the combination of two antiangiogenic agents may lead to improved clinical outcome. These findings could open new perspectives in clinical antitumor therapy.

[Cloning, expression, distribution and subcellular localization of AK078438 gene].

OBJECTIVE: To investigate the molecular cloning, tissue distribution, tumor distribution and the subcellular localization of AK078438 gene. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to amplify the open reading frame of AK078438 gene. Prokaryotic expressing vector and affinity purification were used to get the fusion protein. At mRNA expressing levels semi-quantitative RT-PCR was employed for the investigation of its distribution in normal rat tissues, tumor cell and mesenchymal stem cell (MSC) lines. The eukaryotic expression green fluorescence protein (GFP) fusion protein vector was transfected into cells and identified subcellular localization. RESULTS: The full open reading frame of AK078438 gene with 1065 bp, 355aa were identified. mRNA of the gene distributed in brain, uterus, colon, bone marrow, testis and kidney, but not in stomach, liver, the lung, spleen and heart. Murine colon adenocarcinoma C26, melanoma B16, Lewis lung carcinoma LL/2 and MSC had the gene and mouse myeloma cell line NS-1 and Hepa mouse hepatoma cell line had no the gene. The protein was localized in cytoplasm. CONCLUSION: Protein expression, expressing profile and subcellular localization of AK078438 gene set the basis for further exploration of its functions.

[Nano ultra high performance liquid chromatography coupled with time-of-flight mass spectrometer for detecting low molecular weight polypeptides in zebrafish embryos].

OBJECTIVE: To apply the nano-ultra high performance liquid chromatography coupling with time-of-flight mass spectrometer (nano-UPLC-TOF-MS) to detect the low molecular weight polypeptides in zebrafish embryos. METHODS: Zebrafish embryos were collected 24 hours after fertilization and the outer chorion was removed. And then the yolk was removed with mechanical forces, of which the efficiency was validated by microscope and SDS-PAGE; the low molecular weight polypeptide was extracted with corresponding solution, and followed by separation and detection with nano-UPLC-TOF-MS. RESULTS: Mechanical vortex almost completely removed yolk cells of zebrafish embryos, from which the special solution extracted effectively low molecular weight polypeptides for the later detection. As demonstrated by nano-UPLC-TOF-MS, the polypeptide peak showed between 22 and 24. 5 min, and the m/z ration of most of polypeptides were between 800-1400. CONCLUSION: Nano-OPLC-TOF-MS is effective to detect low molecular weight polypeptides, and low molecular weight peptides definitely exist in zebrafish embryos, of which the relative molecular mass seems to be 800 to 1400, probably consists of about 10 residues.

The Promotion of Erythropoiesis via the Regulation of Reactive Oxygen Species by Lactic Acid.

The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA+ cells. Also, increases in haematocrit and CD71-/Ter119+ erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34+/CD117- cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions.